title: Determination of the GH3.12 protein conformation through HPLC-integrated SAXS measurements combined with X-ray crystallography.
abstract: The combination of protein crystallography and small-angle X-ray scattering (SAXS) provides a powerful method to investigate changes in protein conformation. These complementary structural techniques were used to probe the solution structure of the apo and the ligand-bound forms of the Arabidopsis thaliana acyl acid-amido synthetase GH3.12. This enzyme is part of the extensive GH3 family and plays a critical role in the regulation of plant hormones through the formation of amino-acid-conjugated hormone products via an ATP-dependent reaction mechanism. The enzyme adopts two distinct C-terminal domain orientations with `open' and `closed' active sites. Previous studies suggested that ATP only binds in the open orientation. Here, the X-ray crystal structure of GH3.12 is presented in the closed conformation in complex with the nonhydrolysable ATP analogue AMPCPP and the substrate salicylate. Using on-line HPLC purification combined with SAXS measurements, the most likely apo and ATP-bound protein conformations in solution were determined. These studies demonstrate that the C-terminal domain is flexible in the apo form and favours the closed conformation upon ATP binding. In addition, these data illustrate the efficacy of on-line HPLC purification integrated into the SAXS sample-handling environment to reliably monitor small changes in protein conformation through the collection of aggregate-free and highly redundant data.
authors.affiliation: European Molecular Biology Laboratory, Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble, France.
authors.forename: Adam
authors.initials: A
authors.lastname: Round
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Brown
authors.forename: Romain
authors.initials: R
authors.lastname: Marcellin
authors.forename: Ulrike
authors.initials: U
authors.lastname: Kapp
authors.forename: Corey S
authors.initials: CS
authors.lastname: Westfall
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Jez
authors.forename: Chloe
authors.initials: C
authors.lastname: Zubieta
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D006868:Hydrolysis; D011487:Protein Conformation; D020156:Salicylic Acid / Q000737:chemistry; D053838:Scattering, Small Angle; D013379:Substrate Specificity
chemical_list: D000255:Adenosine Triphosphate; D029681:Arabidopsis Proteins; D020156:Salicylic Acid; C523087:At5g13320 protein, Arabidopsis; C002630:alpha,beta-methyleneadenosine 5'-triphosphate
keywords: GH3 family; acyl acid–amido synthetase; hormone amino-acid conjugates; small-angle X-ray scattering